Novel antigen for use in malaria

ABSTRACT

The present invention provides polypeptides useful as antigens expressed at the pre-erythrocytic stage of the malaria parasite. The antigens can be utilized to induce an immune response and sterile protection against malaria in a mammal by administering the antigens in vaccine formulations or expressing the antigens in DNA or other recombinant protein expression systems delivered as a vaccine formulation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) from U.S.provisional patent application Ser. No. 62/296,464 filed Feb. 17, 2016,the entirety of which is hereby incorporated by reference herein.

BACKGROUND

Despite years of effort, a licensed malaria vaccine is not available.One of the obstacles facing the development of a malaria vaccine is theextensive heterogeneity of many of the malaria vaccine antigens.Potential vaccine antigens that have been evaluated in people thus farhave not elicited a protective immune response.

Malaria kills approximately 863,000 people every year. Although avariety of anti-malarial drugs exist, the cost of these drugs can beprohibitive in the relatively poor areas of the world where malaria isendemic. The widespread use of the most commonly employed drugs has alsoresulted in the expansion of drug-resistant parasites, rendering many ofthese drugs ineffective. In the absence of inexpensive, highly potentdrugs, vaccination represents the most cost-effective way ofsupplementing traditional malaria interventions.

A successful malaria vaccine will need to protect people against a largepopulation of antigenically diverse malaria parasites. A vaccine basedon a single isolate of a single antigen may not be able to elicit animmune response that is broad enough to protect individuals against thisheterogeneous population. One way to potentially enhance the efficacy ofantigen-based vaccines, or any other subunit malaria vaccine, would beto incorporate additional malaria antigens into the vaccine, therebybroadening the immune response elicited by the vaccine.

Malaria vaccine development efforts have focused almost exclusively on ahandful of well-characterized Plasmodium falciparum antigens. Despitededicated work by many researchers on different continents spanning morethan half a century, a successful malaria vaccine remains elusive.Sequencing of the P. falciparum genome has revealed more than fivethousand genes, but has given no indication which of these five thousandgenes will be useful, or how to identify potential vaccine targets.

Malaria is caused by mosquito-borne hematoprotozoan parasites belongingto the genus Plasmodium. Four species of Plasmodium protozoa (P.falciparum, P. vivax, P. ovale and P. malariae) are responsible for thedisease in humans. Others cause disease in animals, such as P. yoeliiand P. berghei. P. falciparum accounts for the majority of infectionsand deaths in humans. Malaria parasites have a life cycle consisting offour separate stages. Each one of these stages is able to inducespecific immune responses directed against the parasite and thecorrespondingly occurring stage-specific antigens, yet naturally inducedmalaria does not protect against reinfection.

Malaria parasites are transmitted to mammals by several species offemale Anopheles mosquitoes. Infected mosquitoes deposit the sporozoiteform of the malaria parasite into the mammalian skin during a bloodmeal, which subsequently invades the bloodstream. Sporozoites remain fora few minutes in the circulation before invading hepatocytes. At thisstage, the parasite is located in the extra-cellular environment and isexposed to antibody attack, mainly directed to the circumsporozoite (CS)protein, a major component of the sporozoite surface. Once sporozoitesinvade hepatocytes, the parasite differentiates, replicates and developsinto a schizont. During this stage, the invading parasite will undergoasexual multiplication, producing up to 20,000 daughter merozoites perinfected hepatocyte cell. During this intra-cellular stage of theparasite, the host immune response includes T lymphocytes, especiallyCD8⁺ T lymphocytes. After 10-14 days of liver infection, thousands ofnewly formed merozoites are released into the bloodstream and invade redblood cells (RBCs), becoming targets of antibody-mediated immuneresponse and T-cell secreted cytokines. After invading the erythrocytes,the merozoites undergo several stages of replication, transforming intotrophozoites, and schizonts, which rupture to produce a new generationof merozoites that subsequently infect new RBCs. This phase(erythrocytic) of the parasite stimulates a strong humoral response thatcan block merozoite invasion of RBCs and usually confers protectionagainst pathology associated with this phase. The erythrocytic stage isassociated with overt clinical disease. A smaller number of trophozoitesmay develop into male or female gametocytes, which are the parasite'ssexual stage. When susceptible mosquitoes ingest gametocytes, thefertilization of these gametes leads to zygote formation and subsequenttransformation into ookinetes, then into oocysts, and finally intosporozoites, which migrate to the salivary gland to complete the cycle.

The two major arms of the pathogen-specific immune response that occurupon entry of the parasite into the body are cellular and humoral. Theone arm, the cellular response, relates to CD8⁺ and CD4⁺ T cells thatparticipate in the immune response. Cytotoxic T lymphocytes (CTLs) areable to specifically kill infected cells that express pathogenicantigens on their surface. CD4+ T cells or T helper cells support thedevelopment of CTLs, produce various cytokines, and also help induce Bcells to divide and produce antibodies specific for the antigens. Duringthe humoral response, B cells specific for a particular antigen becomeactivated, replicate, differentiate and produce antigen-specificantibodies.

Both arms of the immune response are relevant for protection against amalarial infection. When infectious sporozoites travel to the liver andenter the hepatocytes, the sporozoites become intracellular pathogens,spending little time outside the infected cells. At this stage, CD8⁺ Tcells and CD4⁺ T cells are especially important because these T cellsand their cytokine products, such as interferon-γ (IFN-γ), contribute tothe killing of infected host hepatocytes. Elimination of theintracellular liver parasites in the murine malaria model is found to bedependent upon CD8⁺ T cell responses directed against peptides expressedby liver stage parasites. Depletion of CD8⁺ T cells abrogates protectionagainst sporozoite challenge, and adoptive transfer of CD8⁺ T cells tonaïve animals confers protection.

When a malarial infection reaches the erythrocytic stage in whichmerozoites replicate in RBCs, the merozoites are also found circulatingfreely in the bloodstream for a brief period until they invade newerythrocytes. Because the erythrocyte does not express either Class I orII MHC molecules required for cognate interaction with T cells, it isthought that antibody responses against the parasite are most relevantat the blood stage of the parasite lifecycle. In conclusion, a possiblemalaria vaccine approach would be most beneficial if it would induce astrong cellular immune response as well as a strong humoral immuneresponse to tackle the different stages in which the parasite occurs inthe human body.

Current approaches to malaria vaccine development can be classifiedaccording to the different developmental stages of the parasite, asdescribed above. Three types of possible vaccines can be distinguished.The first is pre-erythrocytic vaccines, which are directed againstsporozoites and/or schizont-infected hepatocytes. Historically, thisapproach has been dominated by (CSP)-based strategies. Since thepre-erythrocytic phase of infection is asymptomatic, the goal of apre-erythrocytic vaccine would be to confer sterile immunity, mediatedby humoral and cellular immune response, and thereby prevent latentmalaria infection. This goal has not been met by any known treatment.

The second type of vaccine approach is asexual blood stage vaccines,which are directed against either the infected RBC or the merozoiteitself, are designed to minimize clinical severity or prevent infectionif antibodies prevent merozoites invading erythroctyes. Attempts tocreate such vaccines so far have failed to sufficiently reduce morbidityand mortality or prevent the parasite from entering and/or developing inthe erythrocytes. Transmission-blocking vaccines are designed to hamperthe parasite development in the mosquito host. Attempts to create thistype of vaccine so far have failed to reduce population-wide malariainfection rates.

The final type of vaccine approach is combination malaria vaccines thattarget multiple stages of the parasite life cycle. This approachattempts to develop multi-component and/or multi-stage vaccines.Attempts to create such vaccines so far have failed to effect sufficientprotection. As a result of these failures, there is currently nocommercially available vaccine against malaria.

Immunization of rodents, non-human primates, and humans withradiation-attenuated sporozoites (RAS) has been found to conferprotection against a subsequent challenge with viable sporozoites.However, the expense and the lack of a feasible large-scale culturesystem for the production of irradiated sporozoites, the relativeshort-term efficacy, lack of cross-strain protection, and the need to bedelivered intravenously have been obstacles to the development of suchvaccines.

The CS protein is the only P. falciparum antigen demonstrated to preventmalaria infection when used as the basis of active immunization inhumans against mosquito-borne infection. The protection levels for thisantigen, however, are not high enough to support a viable therapy. Intheory, vaccine protection levels should be above 85% in order to be aviable therapy. With protection lower than that, mutants that are morevirulent may escape in endemic areas. CS antigen-based vaccines havedemonstrated an efficiency of only about 50% and that protection doesnot last more than a year. Nevertheless, this is still the best knownantigen response prior to the present disclosure.

The entire genomic sequence of P. falciparum has been sequenced. SeeBowman et al., Nature, 400: 532-538 (1999); Gardner, et al., Nature,419: 498-511 (2002). Another human malaria parasite, P. vivax, has alsobeen sequenced. See Carlton et al., Nature, 455: 757-763 (2008). Therodent malaria parasite, P. yoelii has also been sequenced. See Carltonet al., Nature, 419: 512-519 (2002). Despite this, however, thedevelopment of efficacious anti-malaria vaccines has been severelyhampered by the inability to identify promising antigens. Sequencing ofthe P. falciparum, P. vivax, and P. yoelii genomes has resulted in theidentification of 5,369, 5,433, and 5,675 genes, respectively. Knowledgeof these sequences alone, however, will not likely result in new vaccineconstructs. Consequently, only 0.2% of the P. falciparum proteome isundergoing clinical testing, and these tests have failed to induce highgrade protection in volunteers.

SUMMARY

The present invention provides polypeptides useful as antigens that areexpressed at both the pre- and erythrocytic stage of the malariaparasite. The antigens can be utilized to induce both cellular andhumoral immune responses against malaria in a mammal by administeringthe antigens in vaccine formulations or expressing the antigens in DNAor other nucleic acid expression systems delivered as a vaccineformulation. In preferred embodiments, the mammal is a human.

In one preferred embodiment, the invention provides an immunogeniccomposition for protecting a mammal against malaria infection, theimmunogenic composition comprising one or more recombinant polypeptidesof SEQ ID NO. 3 or SEQ ID NO. 6, or derivatives thereof in apharmaceutically acceptable carrier. In general, derivatives have atleast 10 contiguous amino acids of and/or 85% identity with thereference sequence. The immunogenic composition can be formed from anisolated or recombinant polypeptide or a carrier virus expressing therecombinant antigen and may be paired with an acceptable adjuvant.

The antigens that are the subject of the present disclosure areidentified by different nomenclatures in different contexts, as isstandard in this art. For convenience, the table below identifies eachantigen by its sequence, as well as the various names and shorthandsused in the prior art and in the disclosure herein:

Shorthand PlasmoDB Identification SEO ID NO. Py E140 PY06306,PY17X_0210400, 1 (amino acid) PYYM_0211900 2 (nucleotide) Pf E140PFA0205w, MAL1P1.31, 3 (amino acid) PF3D7_0104100, XP_001350973 4(nucleotide) Pv E140 PVX_081555, PV081555, 6 (amino acid) PVP01_02106005 (nucleotide) Py falstatin PY17X_0816300, PY03424, PYYM_0816000 PyCSPPY03168, PYYM_0405600 Py E057 PY03396, PY17X_1006600, PYYM_1006600 PyE137 PY05693, PY17X_1006100, PYYM_1006100 Py UIS3 PY03011, PY17X_1402400Pf falstatin, ICP PFI0580C or PF3D7_0911900 7 (amino acid) Pf CSPPFC0210C, MAL3P2.11, 8 (amino acid) PF3D7_0304600 PfUIS3, PF13_0012,PF3D7_1302200 9 (amino acid) ETRAMP13

The invention may comprise a combination of two or more recombinantpolypeptides in a pharmaceutically acceptable carrier, wherein onepolypeptide is SEQ ID NO. 3, SEQ ID NO. 6, or derivatives thereof, andthe other polypeptide is any of the falciparum or vivax orthologs ofPyCSP, Py falstatin, Py UIS3, PY03396, PY05693, PY03424, and PY03011.

The present invention also includes a method of inducing an immuneresponse against malaria in a mammal by administering an immunologicallyeffective amount of a composition comprising one or more polypeptidesencoded by SEQ ID NO. 3 or 6, or derivatives thereof. Alternatively, themethod may include administering one or more priming or boostingimmunizations against malaria, wherein said priming and boostingimmunizations comprise an immunologically effective amount of anrecombinant polypeptide as described. The method of administering thepolypeptide can include use of a suitable expression vector, such as aplasmid, replicating viral vector, or nonreplicating viral vector. Asuitable expression vector can be a DNA plasmid, baculovirus, rVSV,SpyVLPs, alphavirus replicon, adenovirus, poxvirus, adenoassociatedvirus, cytomegalovirus, canine distemper virus, yellow fever virus,retrovirus, RNA replicon, DNA replicon, alphavirus replicon particle,Venezuelan Equine Encephalitis virus, Semliki Forest Virus, or SindbisVirus.

The polypeptides useful as antigens disclosed herein are the firstPlasmodium pre-erythrocytic antigens that can sterilely protect 100% ofsubjects against an infectious P. yoelii sporozoite challenge. Theseresponses are conveniently measured in mice as a proxy for their humanorthologs. Malaria infection, treatment, and immunity has been studiedextensively in both mice and humans, and mouse models are considered astandard indicator of malaria vaccine efficacy in human and othermammalian subjects. The PY06306 antigen disclosed herein alone protects71% to 100% of CD1 mice against malaria and in addition induces animmune response capable of delaying the parasite onset in the blood ofremaining non-protected mice. Overall, 83% (384/461) ofPY06306-immunized mice were protected from malaria infection. Thisprotection is reported for both outbred (CD1) and inbred (BABB/c)strains of mice, using a rigorous 300- and 100-sporozoite challenge,respectively, and efficacy assessment as sterile protection. Theefficacy of the antigen disclosed herein, in light of the relationshipsamong murine, primate, and human malaria immune responses disclosedherein, and standard indicators of vaccine efficacy, presents apolypeptide for inducing an immune response against malaria in a mammal.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the protection results for a matrix experiment in whichfourteen CD1 outbred mice per group were immunized in a prime-boostregimen with a combination of DNA and Human Adenovirus type 5 (Ad5)vectors that express PY03396, PY05693, PY06306, PY00232 and PyCelTOS.Positive control mice were immunized with DNA and Ad5 vectors thatexpress PyCSP. Negative control mice were immunized with 4X relativeamount of DNA and Ad5 vector that do not express P. yoelii antigen andnaïve mice. Gray and black bars indicate antigen combination groups withand without PyCSP, respectively. Hatched and checkered bars representPyCSP and naïve groups, respectively. The mice were challenged with 300P. yoelii sporozoites and evaluated for parasitaemia by examiningGiemsa-stained blood smears up to 14 days post challenge. Numbers atbottom denote number of sterile protected mice per total challenged micein each group.

FIG. 2 shows a matrix deconvolution of the experiment evaluating PY06306and other antigens shown illustrated in FIG. 1. Fourteen CD1 outbredmice per group were immunized in a prime-boost regimen comprising of DNAand Adenovirus type 5 (Ad5) vectors that express PY03396, PY05693,PY06306, PY03424 and PY03011. Positive control mice were immunized withDNA and Ad5 vectors that express PyCSP. Negative control mice wereimmunized with 4X relative amount of DNA and Ad5 vectors that do notexpress P. yoelii antigen. Gray and black bars indicate antigencombination groups with and without PyCSP, respectively. Hatched andcheckered bars represent PyCSP and null-immunized mice, respectively.The mice were challenged with 300 P. yoelii sporozoites and evaluatedfor parasitaemia by examining Giemsa-stained blood smears up to 17 dayspost challenge. Numbers at bottom denote number of sterile protectedmice per total challenged mice in each group.

FIG. 3 shows a Kaplan-Meier curve depicting the percentage of protectedmice for the time to parasitemia after challenge. Data extracted andanalyzed from matrix deconvolution experiment 2. Closed circles indicateCD1 mice immunized with PY06306 antigen alone, Symbols Xs, squares andtriangles indicate PyCSP, 4X Null and Naïve mice, respectively. The micewere challenged with 300 P. yoelii sporozoites and evaluated forparasitaemia by examining Giemsa-stained blood smears up to 14 (PyCSP,4X Null and Naïve) or 17 (PY06306) days post challenge.

FIG. 4 shows antibody responses for the matrix deconvolution experiment.Endpoint immunofluorescence assay (IFA) titers were measured on P.yoelii sporozoite and blood stage parasites. Sera collected one weekafter Adeno 5 boost was pooled per group of antigen combination andassayed for reactivity on air-dried parasites. Black and gray barsindicate sporozoite and blood stages reactivity, respectively. Positivecontrol antibodies were NYS1 and NYLS3 monoclonal antibodies,respectively. Sera from 4X null and naïve animals were negative.

FIG. 5 shows antibody titers of protected and non-protected mice for thematrix deconvolution Experiment shown in FIG. 4. EndpointImmunofluorescence (IFA) titers were measured against P. yoeliisporozoite for individual mice for six PY06306 (E140)-containing groupsof mice. One group from matrix experiment 2 (Mx2); E140, E137, E057combination (closed circles) and five groups from matrix deconvolutionexperiment 2 (MDx2); E140, E137, E057 combination (closed squares), E140alone (closed diamonds), E140, E137 combination (closed stars), E140,E057 combination (closed triangles), and E140, E137, E057, PY3424combination (closed asterisks). All protected mice are displayed byclosed symbols and all non-protected by the X symbol. Mann-Whitneynon-parametric test indicates statistical significance;**, p<0.005and***, p=0.001.

FIG. 6 shows continued protection at 11 weeks for the deconvolutionstudy shown in FIG. 2. Sterilely protected mice were rested for 11 weeksand then challenged with 200 P. yoelii sporozoites. Protection wasmeasured by examining Giemsa-stained blood smears up to 17 days postchallenge.

FIG. 7 shows the PY06306 (Py E140) antigen homology among Plasmodiumspp, including Pf (human P. falciparum), Pv (human P. vivax), Pc (rodentP. chabaudi), Py (rodent P. yoelii), Pb (rodent P. berghei), Pk (primateP. knowlesi), Pr (primate P. rhodiani), and Pg (primate P. gaboni).

FIG. 8 shows the PY06306 (Pf E140) (PFA0205w or MAL1P1.31 orPF3D7_0104100) amino acid conservation among various Pf parasitestrains. These parasites were collected from a variety of countries indifferent continents. The highest (99%) and the lowest (92%) homologyare highlighted.

FIG. 9 shows the results of an in vivo T cell depletion experiment inmice. CD1 outbred mice were immunized with PY06306 DNA and boosted withAdeno 5 vaccines, CD4⁺, CD8⁺, CD4⁺/CD8⁺ T cells depleted (black bars)before and after challenge with 300 P. yoelii sporozoites. Rat Ig and nodepletion groups were used as positive controls. Groups ofnull-immunized mice (gray bars) were also depleted the same way and usedas negative controls. PyCSP (diagonal bar) and Naïve (stripe bar) wereexperimental positive and negative controls. Arrows indicate the type ofdepletion and the number of mice sterile protected out of the numberimmunized. Challenged mice were evaluated for parasitaemia by examiningGiemsa-stained blood smears up to 19 days post challenge.

FIGS. 10A and 10B shows sera transfer studies in CD1 and BALB/c mice. InFIG. 10A, groups of 14 BALB/c mice were either immunized with DNA/Adenovirus 5 encoding PY06306 (solid black line) and PyCSP (solid gray line).Sera from immunized and non-challenged mice were collected andtransferred 24 and 6 hours before challenge to naïve recipient mice;PY06306 (dotted black line) and PyCSP (dotted gray line). Afterchallenge with 300 P. yoelii sporozoites, mice were monitored forparasitaemia for 17 days. In FIG. 10B, groups of 14 CD1 mice were eitherimmunized with DNA/Adeno virus 5 encoding PY06306 (solid black line) andPyCSP (solid gray line). Sera from immunized and non-challenged micewere collected and transferred 24 and 6 hours before challenge to naiverecipient mice; PY06306 (dotted black line) and PyCSP (dotted grayline). After challenge with 100 P. yoelii sporozoites, mice weremonitored for parasitaemia for 17 days. Percentage of sterilelyprotected mice for each group is shown in the legend box.

FIG. 11 shows PY06306 protection against a blood stage challenge,Fourteen CD1 mice per group were immunized with a dose of DNA andboosted with Adenovirus 5 expressing PY06306 (black bar),PY06306+PyFalstatin (gray bar), and PyFalstatin alone). Null-immunizedand naïve were used as negative control groups of mice. PyFalstating isalso known as PY03424. All mice were challenged with 10,000 infected P.yoelii-infected erythrocytes and parasitaemia monitored for 17 daysafter challenge by Giemsa-stained thin smears.

FIG. 12 shows protection with mammalian codon-optimized Adenovirus 5 ina chart comparing native (na) and codon-optimized (co) PY06306 and routeof immunizations. CD1 mice (14 per group) were primed with a co E140 DNAand boosted with either native PY06306 Adeno 5 (black bars) or mammalianco PY06306 Adeno 5 (gray bars). Both Adeno 5 constructs wereadministered intramuscular (IM) in decreasing doses from 10̂10, 10̂9, 10̂8,and 10̂7 PU. Two additional groups of mice were boosted with Ad5administered either subcutaneously (SC) or intravenously (IV). Twoadditional mice groups were not primed with DNA vaccine and insteadimmunized with a single IM dose of either na or co PY06306 Ad5 two weeksbefore challenge. Null-immunized (stripe bar) and Naïve (checkered bar)mice are negative controls. All mice were challenge with 300 P. yoeliisporozoites, parasitaemia were monitored over 18 days by thin bloodsmears stained with Giemsa.

FIG. 13 shows that Pf E140 (PFA0205w or MAL1P1.31 or PF3D7_0104100) isimmunogenic in mice. IFA titers induced by PFA0205w vaccines. Both CD1and BALB/c mice were immunized with PFA0205w (PIE140) vaccines reagents:DNA vaccine in VR1020-DV plasmid, Adenovirus 5, and full lengthrecombinant protein expressed by the wheat germ system as GST and 6xHisfusions. Recombinant proteins were emulsified in Montanide ISA 720adjuvant and immunized SC as 5 μg/dose. Immunofluorescence (IFA) titerswere measured against both P. falciparum sporozoites and a mixture ofseveral of blood stages.

FIG. 14 shows that the P. falciparum E140 (PFA0205w) is naturallyimmunogenic in humans. T cell responses to PFA0205w (PfE140 orPF3D7_0104100) by P. falciparum radiation attenuated sporozoites(RAS)-immunized human subjects. PBMCs were stimulated with overlapping15 mer peptide PFA0205w pools A for 21 h with brefeldin A and stainedfor viability, phenotypic (CD14, CD19, CD3, CD4, and CD8), andintracellular functional markers (including IFN-γ and CD154). Thebackground subtracted frequencies of CD4⁺ T cells producing IFN-γ andintracellular CD154 (A) and CD8⁺ T cells producing IFN-γ(B) are shown.Positive responses for PFA0205w pool A (filled symbols) in bothexperiments were identified as those exceeding two standard deviationsfrom the average of the negative control (DMSO stimulated) samples.

FIG. 15 shows that PVX_081555 (PvE140) is relatively abundant in P.vivax sporozoites. 256 P. vivax sporozoite proteins sequenced usingmulti-dimensional-protein-identification-technology (MudPIT) weregraphed based on their relative abundance defined by their quantitativevalue. The positions of P. vivax circumsporozoite protein and P. vivaxE140 (PVX 081555) are indicated in the graph with a black arrow.

DETAILED DESCRIPTION

The inventor has determined that pre-erythrocytic proteins are criticalin conferring protective immunity against malaria. Despite therelatively large number of malaria genes that have been identified,following sequencing of the malaria parasite genome, identification ofvaccine candidates has been hampered, to a great extent, by therelatively complex life-cycle of malaria parasite. Furthermore, manygenes of the malaria parasite are poorly defined, antigenically, as wellas functionally.

Against this backdrop, the inventor decided to undertake high-throughputscreening of antigens encoded by numerous genes in order to ascertainpotential protective responses. The inventor developed a novel strategyfor identifying and testing potential malaria antigens that overcame thedifficulties experienced in the prior art. This novel approach includedidentifying certain traits that the inventor determined would beindicative of potential human vaccine candidates. The inventor thencompiled a list of 146 P. yoelii orthologs of P. falciparum genes thatwere believed to possess these traits. The inventor then designedcloning primers, and conceived of a strategy for cloning the genes andscreening by transfection ELISpot. The transfection ELISpot involvedtransfecting an A20 cell line with the VR1020 vaccine constructs,expressing the antigen, and using these transfected cells to presentantigens in the ELISpot assay. This use of ELISpot was a novel strategyfor screening antigens. Priority antigens were identified from a largepanel of P. falciparum proteins. The priority antigens were evaluatedbased on a number of criteria judged by the inventor to be relevant toprotection against malaria. One such criterion was selecting antigensthat are expressed in the sporozoite and liver stages of the malariaparasite; i.e. pre-erythrocytic antigens. Certain antigens among thoseselected based on this criterion showed protective responses in micethat indicated that orthologs of those genes in humans would encodehuman antigens useful as potential vaccine formulations. One gene inparticular, PY06306, later curated as PY17X_0210400, which is thesubject of this disclosure, surprisingly showed dramatic and consistentprotection responses indicating that gene as encoding an antigen forwhich orthologs would be useful as a leading vaccine formulation.

The sequence documented for the PY06306 gene, however, was only partial(479 aa) and originated from the early genome annotation. In order toperform the protection experiments disclosed herein with the full-lengthantigen (816 aa), the inventor needed to re-clone the gene. A similarsituation occurred with the P.falciparum (human homolog), which alsoneeded to be re-cloned from what was known in the art. The sequencesdisclosed in the listing provided herein, used in all of the examples,and reflected in all of the data examples conform to the inventor'scorrected version of the gene, rather than what was previously believedin the art to be the relevant sequence.

The invention relates to DNA and amino acid sequences encodingrecombinant Plasmodium falciparum and Plasmodium vivax proteins.Specifically, the invention relates to a highly protectivepre-erythtrocytic Plasmodium yoelii and its P. falciparum and P. vivaxortholog antigens for use in a malaria vaccine. The relevant sequencescan be utilized to express the encoded proteins for use as subunitimmunogenic antigens or can be incorporated into vectors suitable for invivo expression in a host in order to induce an immunogenic response.The antigens can be utilized in combination or singly in immunogenicformulations.

In one embodiment, the immunogenic composition is a DNA-based vaccine.DNA was found to be a viable platform for delivering the immunogeniccompositions of the present disclosure. A DNA-based vaccine can bedelivered by recombinant viruses, such as Modified Vaccinia Ankara (MVA)attenuated poxvirus, Vesicular Stomatitis Virus (VSV), or GC46 (gorillaadenovirus) viruses. Other human Adenovirus alternatives like these canalso be used, such as baculovirus.

In another embodiment, the composition comprises immunogenic proteins.In this embodiment, the proteins can be produced by first inserting theDNA encoding the proteins in suitable expression systems. These include,for example, Adenoviral based systems, a poxvirus based system, or a DNAplasmid system. The expressed and purified proteins can then beadministered in one or multiple doses to a mammal, such as humans. Inthis embodiment, the purified proteins can be expressed individually orDNA encoding specific proteins can be recombinantly associated to form asingle immunogenic composition. These immunogenic compositions can thenbe administered in one or multiple doses to induce an immunogenicresponse.

One embodiment of the invention relates to recombinant polypeptidesexpressed as full-length or fragments by heterologous expressionsystems. Examples of such systems are: Escherichia coli, yeast(Saccharomyces cerevisiae or Pichia pastoris), mammalian cells (HEK293or CHO cells), baculovirus-infected insect cells, and Drosophila S2stable cells. The recombinant proteins can be incorporated inimmunogenic formulations in order to induce an immune response. In thisembodiment, the polypeptides can be incorporated singly or incombination. The immunogenic compositions of the invention can alsoinclude adjuvants to improve or enhance the immune response elicited bythe polypeptides. Suitable adjuvants include ALFQ, a non-toxicformulation comprising a monophosphoryl lipid A-containing liposomecomposition with saponin.

Adjuvants have traditionally been broadly classified into two majorclasses according to their component sources, physiochemical propertiesor mechanisms of action, namely: (i) immunostimulants such as TLRligands, cytokines, saponins and bacterial exotoxins that directly acton the immune system to increase responses to antigens and (ii) vehiclessuch as mineral salts, emulsions, liposomes, virosomes and biodegradablepolymer microspheres that present vaccine antigens and co-administeredimmununostimulants to the immune system in an optimal manner. In recentyears it has become apparent that many of these vehicles also have adirect effect on the immune system and can be consideredimmunostimulants.

Examples of acceptable adjuvants for inclusion with a malaria vaccineinclude Army Liposome Formulation (ALF) derivatives such as ALF, ALFA(plus aluminum), and ALFQ (plus QS21). Other options include a lipid Aderivative and a saponin in a liposome formulation, such as QS21 and3D-monophosphoryl lipid A (a non-toxic derivative oflipopolysaccharide), other immunostimulants that are similar instructure to LPS, MPL, or 3D-MPL, acylated monosaccharides, saponinderivatives (Quil-A, ISCOM, QS-21, AS02 and AS01), soluble triterpeneglycosides, Toll-like receptor 4 (TLR4) agonists, montanides (ISA51,ISA720), immunostimulatory oligonucleotides, and imidazoquinolines.Adjuvants may be prepared in cholesterol-containing liposome carriers.

As used herein, the term “polypeptide” refers to a polymer of aminoacids and does not refer to a specific length of the product. Proteinsare included within the definition of polypeptides. The term “mer,” inconjunction with a number, such as 15-mer, refers to the length of apolypeptide in numbers of amino acids.

As used herein, the proteins may be prepared for inclusion of aneffective amount of one or more polypeptides described herein into animmunogenic composition by first expressing the appropriate genefragments by molecular methods, expression from plasmids or otherexpression systems such as viral systems and then isolated. A furtheraspect of the invention is the ability of the proteins to induce anhumoral and/or T-cell immune response.

An embodiment of the invention is the incorporation of DNA encoding thepolypeptides in vector expression systems, wherein the system permitsexpression of one or more polypeptides in mammalian host cells, such asin humans to induce an immune response. The expression systems can beDNA plasmids or viral systems. Methods for preparing and administering aDNA vaccine expressing Plasmodium proteins are well known in the art.

In another embodiment, derivatives of the proteins can be used inimmunogenic compositions. In a variant of this embodiment, theimmunogenic derivatives of the P. falciparum and P. vivax proteinsinclude at least 10 contiguous amino acids of an amino acid sequence ofa full length polypeptide comprising an amino acid sequence disclosedherein. Immunogenic derivatives of the polypeptides may be prepared byexpression of the appropriate gene fragments or by other methods such asby peptide synthesis. Additionally, derivatives may be a fusionpolypeptide containing additional sequence encoding one or more epitopesof the P. falciparum polypeptides disclosed herein. In theseembodiments, the proteins can be directly incorporated in immunogenicformulations or expressed from DNA plasmids or viral expression systems.

In some embodiments, the P. falciparum and P. vivax polypeptides includeimmunogenic derivatives with more than 80% amino acid sequence identityto the sequences disclosed herein. In this context, the term “identity”refers to two or more sequences or subsequences that are the same orhave a specified percentage of amino acid residues that are the same,when aligned for maximum correspondence. Where sequences differ inconservative substitutions, i.e., substitution of residues withidentical properties, the percent sequence identity may be adjustedupwards to correct for the conservative nature of the substitution.

When the compositions are prepared for administration, they arepreferably combined with a pharmaceutically acceptable carrier, diluentor excipient to form a pharmaceutical formulation, or unit dosage form.A “pharmaceutically acceptable carrier” is a carrier, diluent,excipient, and/or salt that is compatible with the other ingredients ofthe formulation, and not deleterious to the recipient thereof. Theactive ingredient for administration may be present as a dry powder oras granules; as a solution, a suspension or an emulsion. The compositionexists as dry powder prior to reconstitution in a liquid carrier.

Pharmaceutical formulations containing the immunogenic compositions ofthe invention can be prepared by procedures known in the art using wellknown and readily available ingredients. The therapeutic agents of theinvention can also be formulated as solutions appropriate for parenteraladministration, for instance by intramuscular, subcutaneous orintravenous routes. The pharmaceutical formulations of the therapeuticagents of the invention can also take the form of an aqueous oranhydrous solution or dispersion, or alternatively the form of anemulsion or suspension.

Thus, the immunogenic composition may be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose form in ampules,pre-filled syringes, small volume infusion containers or in multi-dosecontainers with an added preservative. The composition is suitable forinjection intravenously, subcutaneously, or intramuscularly. The activeingredients may take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing, and/or dispersing agents. Alternatively, theactive ingredients may be in powder form, obtained by aseptic isolationof sterile solid or by lyophilization from solution, for constitutionwith a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

Additionally, the immunogenic composition may contain formulatory agentsthat do not occur naturally in the cellular environment in which thepeptide is expressed. Such formulatory agents include any surfactants,diluents, solubilizers, emulsifiers, buffers, thickeners, preservatives,detergents, adjuvants, excipients, and antimicrobials that do notnaturally occur in the cellular environment in which the peptide isexpressed, but nonetheless serve to artificially enhance thebioavailability, effectiveness, delivery, storage, administration,absorption, stability, safety, or function of the peptide in theimmunogenic composition before, after, or during administration to amammal.

Alternately, the immunogenic composition may be provided as a drypowder. A dry powder composition may be prepared by freeze drying, spraydrying, and freeze spray drying a solution or suspension containing thepolypeptides described herein, and may further optionally includemilling or lyophilization with milling. The dry powder may be suitablefor direct administration to a patient, such as through inhalation orcapsule ingestion, or may be suitable for suspension or reconstitutionin a fluid carrier. Dry powder formulations may include physiologicallyacceptable carrier powders, such as excipients, dispersants,stabilizers, humectants, anti-caking agents, or other additives.

The immunogenic compositions of the present invention, both dry powderand fluid embodiments, may include, as optional ingredients,pharmaceutically acceptable carriers, diluents, solubilizing, oremulsifying agents, and salts of the type that are well-known in theart. Specific non-limiting examples of the carriers and/or diluents thatare useful in formulations of the present composition include water andphysiologically acceptable buffered saline solutions, such as phosphatebuffered saline solutions pH 7.0-8.0. The composition of the presentdisclosure may also comprise combinations of other agents such asdiluents, which may include water, saline, glycerol or other suitablealcohols, wetting or emulsifying agents; buffering agents; thickeningagents for example cellulose or cellulose derivatives; preservatives;detergents; antimicrobial agents; and the like.

Where the immunogenic composition is used as a vaccine, the compositioncomprises an immunologically effective amount of the peptides describedherein. An “immunologically effective amount” of an antigen is an amountthat when administered to an individual, either in a single dose or in aseries of doses, is effective for treatment or prevention of malariainfection. This amount will vary depending upon the health and physicalcondition of the individual to be treated and on the antigen.Determination of an effective amount of an immunogenic or vaccinecomposition for administration to an organism is well within thecapabilities of those skilled in the art.

A composition according to the invention may be for oral, systemic,parenteral, topical, mucosal, intramuscular, intravenous,intraperitoneal, intradermal, subcutaneous, intranasal, intravaginal,intrarectal, transdermal, sublingual, inhalation or aerosoladministration. The composition may be arranged to be administered as asingle dose or as part of a multiple dose schedule. Multiple doses maybe administered as a primary immunization followed by one or morebooster immunizations. The primary immunization may include a singleformulation such as a virus (GC46) or DNA vaccine, followed by one ormore booster immunizations with single or multiple formulations such asanother virus (such as MVA) or recombinant protein. Suitable timingsbetween priming and boosting immunizations can be routinely determined.A composition according to the present disclosure may be used inisolation, or it may be combined with one or more other immunogenic orvaccine compositions, and/or with one or more other therapeutic regimes.

The present disclosure thus provides a method of protecting a human ornon-human mammal from the effects of malarial infection comprisingadministering to the human or non-human mammal a composition describedherein. The composition may be a vaccine. The disclosure furtherprovides a method for raising an immune response in a human or non-humanmammal comprising administering a pharmaceutical composition describedherein to the human or non-human mammal. The immune response ispreferably protective. The method may raise a booster response in apatient that has already been primed. The immune response may beprophylactic or therapeutic.

EXAMPLES Example 1 Identification of E 140

A novel, highly protective pre-erythrocytic (PE) Plasmodium yoelii (Py)antigen, human orthologs for which are identified for use in a humanmalaria vaccine. This antigen is identified as PlasmoDB ID 10: PY06306,or PY17X_0210400, PYYM_0211900 or ID: 2121.m00052, depending on thenomenclature used. The antigen is also referred to as E140 or Py E140 inlaboratory testing disclosed herein as a shorthand. The novel antigen ishighly expressed in the sporozoite, liver, and blood stages of theparasite, and induces CD8⁺ T cell responses in mice immunized with theP. yoelii radiation-attenuated sporozoites (RAS). It generates strongantibody and cellular responses upon antigen-specific vaccineimmunizations and sterilely protects between 71%-100% alone and incombination with other antigens of mice from an infectious P. yoeliisporozoite and blood stage challenges. First, P. yoelii pre-erythrocyticantigens were screened for their reactivity to T cells fromRAS-immunized mice as a platform for identifying antigens for vaccinedevelopment. This process involved identifying, cloning, generating DNAplasmid (VR1020), screening, and evaluating Py antigens for ability toprotect mice. It is well recognized that mouse models are a predictorfor success with human orthologs. The gene encoding the PY06306 antigenwas identified as a pre-erythrocytic target for vaccine development, andthe partial gene was cloned. Experiments then determined that theprotein could recall cytokine (IFN-γ) responses from splenocytesgenerated in mice immunized with the P. yoelii RAS. This data providedstrong evidence that the PY06306 antigen was involved in the RAS immuneresponse and protection, therefore demonstrating pre-erythrocyticvaccine value in humans.

Example 2 Confirming E140 Protection

Two vaccine reagents were made expressing the PY06306 antigen forprotection studies in mice. These reagents were generated with thefull-length gene: DNA vaccine in the VR1020 plasmid (PY06306-E140) andadenovirus serotype 5 (AdE1(t.PY06306)E3(10X)E4(TIS1)). The evidence forvaccine potential of the PY06306 antigen is shown in two separate animalmatrix studies, intended to assess the ability of the antigen to inducean immune response capable of sterilely protecting mice from aninfectious Py sporozoite challenge. The sterile protection was measuredby the absence of parasites in the blood of mice examined up to 14 or 17days post sporozoite challenge. Outbred CD1 mice were immunized with aregimen consisting of a prime with DNA vaccine (100 μg, IM) and a boostwith adenovirus serotype 5 constructs (10¹⁰ PU, IM) 6 weeks later. A3-antigen combination strategy (named matrix) was adopted to test thePY06306 antigen plus other new Py pre-erythrocytic antigens with andwithout P. yoelii circumsporozoite protein (PyCSP).

The first matrix animal study shown in FIG. 1 revealed twoPY06306-containing antigen combinations (groups) yielding significantprotection. The first combination induced 64% and 86% sterile protectionalone and with PyCSP, respectively. The antigen components of this firstcombination were E140 (PY06306), E137 (PY05693) and E057 (PY03396). The86% protection of the 3-antigen mixture combined with PyCSP was twice ashigh as the PyCSP alone group (43%), indicating a significantenhancement in the efficacy of this gold standard vaccine. The second3-antigen combination produced 14% and 71% sterile protection alone andwith PyCSP, respectively. This second combination consisted of E140(PY06306) combined with two additional antigens with vaccine potential:Py325 (PY00232) and PyCeITOS (PY17X_1434600). Any or all of these fiveantigens (PY06306, PY05693, PY03396, PY325, and PyCeITOS) contributes tothe protection shown in the corresponding figure; however, PY06306 wasthe only antigen common to all three antigen combinations, thusrequiring a second experiment for the deconvolution of these antigencombinations.

Example 3 Sporozoite Challenge

A second study (Matrix Deconvolution Experiment 2) was designed toevaluate several antigen combinations having the PY063Q6 as the commondenominator antigen. The experimental format and immunizations followedthe same regimen as described for the first matrix experiment. FIGS. 1and 2 show the markedly high efficacy for all antigen combinations thatinclude the PY06306 (E140) antigen, ranging from 71% to 100% of the miceprotected. Overall, 89% (137/154) of PY06306-immunized mice wereprotected from malaria infection. The PY06306 vaccine alone yielded 71%protection, significantly higher compared to 36% for the PyCSP alonegroup. Furthermore, there was a substantial delay in the onset ofparasitaemia of non-protected mice as shown in FIG. 3. Detailed analysisof blood smears data from the PY06306-immunized group shows that threeof the four non-protected mice became malaria positive on days 7, 10 and12 after sporozoite challenge. This is significant when compared to theparasitaemia onset of the PyCSP, 4X Null, and Naive groups, in which allnonprotected mice became malaria positive by day 5 post sporozoitechallenge.

Example 4 Antibody Titers

The PY06306 antigen induces high antibody titers to P. yoelii sporozoitestages and low antibody levels to blood stages depending on theindividual mouse. This evidence is shown in FIG. 4 (PY06306 group)listing immune fluorescence (IFA) antibody titers to both sporozoite andblood stage parasites measured in pooled sera from mice in the MatrixDeconvolution Experiment 2. In summary, anti-sporozoite antibodies weredetected in all groups immunized with PY06306, including combinations,which supports the immunogenicity of PY06306 antigen. Titers range from1:5,120 to 1:20,480. Antibodies induced by the P. yoelii PY06306immunization cross-reacts to P. berghei sporozoites. The detection ofhigh antibody titers (1:5,120) in mice immunized with PY06306 alonedemonstrates that the PY06306 antigen induces antibodies to sporozoites.

Two important observations based on a review of the data are: (i) theabsence of protection (0%) and the lack of antibody response for thegroup of antigens without PY06306 (PY03396 and PY05693) in FIG. 2. Thisconfirms that the PY06306 is the main, if not the only component ofthese combinations inducing protection. The other (ii) is theanti-sporozoite antibody response induced specifically by the PY06306antigen. The comparison of the anti-sporozoite IFA titers for theprotected versus non-protected mice strongly indicates that theantibodies detected in these mice correlate with the protection outcome.All protection studies were performed under animal protocols D02-09 and14-IDD-13. The results of the protection studies validate the role ofPY06306 orthologs as valuable components for a malaria vaccine.

Example 5 Spleen and Liver Analysis

Further studies confirmed that in spleen, >10% CD8+ T cells expressingIFNγ and lower (<0.6%) CD4+ T cells in PY06306-immunized mice. A rangeof 5% to 16.2% in liver was observed. High efficacy of protectioncontinued 11 weeks after a second sporozoite challenge. The T celldepletion indicates that high levels of E 140-specific T cells are notrequired for protection in mice. Additionally, PY06306 immunizationinduces high levels of CD8+ T cells expressing IFNγ in the spleen liver.Anti-PY06306 sera transfer to both CD1 and BALB/c mice significantlydelayed the onset of parasitemia. E140-sera recipient mice also hadsignificantly lower IFA titers compared to protected mice immunized withPY06306. PY06306 sera collected prior to sporozoite challenge reacts tosporozoites only. However, after challenge some protected mice developedantibodies positive to blood stage by IFA.

PY06306 sterilely protects up to 100% of CD1 and BALB/c mice from ablood stage challenge (FIG. 11). Immunization with PY06306 preventsblood infection and delays onset of detectable parasitemia in 88%(30/34) of non-protected mice. Additionally, transfer of anti-PY06306antibodies to naïve mice significantly delays infection. High levels ofCD8+ T cells expressing IFNγ in are found in spleens and livers ofPY06306-immunized mice. Depletion did not reduce sterile protection.PY06306-specific IFA antibody titers correlate with protection.

Example 6 In Vivo T Cell Depletion

FIG. 9 shows the results of a study on in vivo t-cell depletion. Severalgroups of outbred CD1 mice were immunized. T cell depletions wereperformed by injection of T cell-specific monoclonal antibodiesfollowing standard protocols. Mice were then challenged with 300 P.yoelii sporozoites and protection assessed by the absence of parasitesin thin blood smears up to 19 days after challenge. AllPY06306-immunized mice that had their T cells depleted were protected,confirming that both CD4+and CD8+T cells are not required for thePY06306 protection. One non-protected mouse from the CD4/CD8 group hadmalaria detected in the blood 13 days post sporozoite challenge whileall other mice had positive smears on day 5. A total of 68 protectedmice out 70 were immunized, a 97% overall efficacy. This study confirmedthe surprising mechanism that protection induced by a pre-erythrocyticantigen against a sporozoite challenge does not rely on T cells.

Example 7 Sera Transfer Studies

FIGS. 10A and 10B shows sera transfer studies in CD1 and BALB/c mice.This study confirmed the role of antibodies in the protection induced byPY06306 (E140). The study design followed standard sera transferprotocols, where sera from PY06306-immunized CD1 and BALB/c mice wereharvested, transferred to naive animals (1:1 ratio), and then challengedwith P. yoelii sporozoites. Sera transfers took place over 2 days; 24hours and 6 hours before the sporozoite challenge. The protectionresults are shown in FIG. 10A for CD1 mice and FIG. 10B for BALB/c mice.FIGS. 10A and 10B show that no sterile protection was transferred withsera (7% (1 out 14) of CD1 and 0% (0 out 14) of BALB/c) from miceimmunized with PY06306 vaccine. There was a statistically significantdelay in the onset of parasitemia on all non-protected mice from thePY06306 sera recipient (dotted line) as compared to any other group inthe same study (Mantel-Cox ***, p=0.0001). This confirms that theanti-PY06306 antibodies have an effective impact on the parasitedevelopment in the blood play a role in the protection. Significantlylower antibody titers in the recipient CD1 (1:2,560) and BALB/c (1:575)mice and compared to the donor CD1 (1:7,994) and BALB/c (1:18:549) miceexplain why these mice were not protected from the challenge.

Example 8 Detection of PY06306-Specific CD8 T Cells in Spleen and Liver

PY06306-specific CD8 T cells are found in the spleens and livers ofPY06306-immunized and naïve mice. Due to the fact that PY06306 is alarge molecule, 15 mer overlapping peptides were divided into two poolsspanning the entire protien; Pool A containing peptides from theN-terminal and Pool B from the C-terminal of PY06306. T cells weremeasured by flow cytometry gated for CD8+cells expressing Interferongamma (IFNγ) and expressed as a percentage of the total T cellpopulation. The data shows that only peptides from Pool A were able torecall IFNγ CD8 cells confirming that PY06306 T cell epitopes are likelyrestricted to the N-terminal of the antigen. Very high levels of CD8+Tcells expressing IFNγ were detected for both spleens (average 18%) andlivers (average 11%) of PY06306-immunized mice. For intracellularcytokine staining, splenocytes and liver-resident T cells were preparedfrom PY06306- and Null-immunized mice using standard protocols, followedby stimulation for six hours with a final concentration of 2 μg/ml ofPY06306 (E140) peptide pools A and B. Data were acquired using a LSRIIflow cytometer (BD Biosciences) and analyzed using FlowJo (Tree StarInc.).

Example 9 PY06306 Induces Protection in BALB/c Mice

PY06306 antigen effectively protects BAB/c strains of mice against asporozoite challenge. Fourteen BALB/c mice per group were immunized witha dose of DNA and boosted with Adenovirus 5 encoding PY06306,PY06306+PyCSP, and PyCSP. Null-immunized and naïve were used as negativecontrol groups of mice. All mice were challenged with 100 infectious P.yoelii sporozoites and parasitaemia monitored for 17 days afterchallenge by Giemsa-stained thin smears. Upon challenge all (100%)PY06306-immunized mice were sterilely protected (PY06306 andPY06306+PyCSP) whereas 57% of PyCSP were protected. Thus PY06306 canprotect an inbred strain of mice, and mixing with PyCSP antigen does notinhibit the PY06306 protection.

Example 10 PY06306 Induces Protection Against a Blood Stage Challenge

FIG. 11 shows PY06306 protection against a blood stage challenge.PY06306 antigen alone and in combination with PyFalstatin protects miceagainst a stringent challenge with 10,000 blood stage parasites. In thisstudy, mice immunized with PY06306 alone and in combination withPyFalstatin and challenged with P. yoelii-infected erythrocytes. Bothgroups of mice were 100% sterilely protected (black and gray bars).PyFalstatin antigen is also known as PY03424. The protection against ablood stage challenge provides a second level of defense induced by thePY06306 vaccine, a valuable feature for a malaria vaccine.

Example 11 Protection with Lower and Single Dose of Codon-OptimizedPY06306 Ad5

FIG. 12 shows protection with codon-optimized Adenovirus 5. This studyevaluated an Adenovirus 5 construct made with codon-optimized (co)PY06306 gene designed for expression in mammalian cells. The change inthe PY06306 native codon sequence did not alter the amino acid sequenceexpressed by the Ad5 virus. A study examined and compared the in vitroexpression of the PY06306 protein expressed by the native (na) andcodon-optimized (co) Adenovirus 5 constructs. After probing with mousepolyclonal sera, the coPY06306 Ad5 expresses much higher levels ofPY06306 protein compared to the native construct. In the first groups ofmice, the boosting dose was titrated for both native (na) andcodon-optimized PY06306 Ad5 ranging from 10̂10, 10̂9, 10̂8 and 10̂7 PU perdose. All mice in these eight groups were primed with the same coPY06306DNA vaccine dose (100 μg) and boosted with varying doses of eithernaPY06306 (black bars) or coPY06306 (gray bars) Ad5 constructintramuscular (IM). The overall efficacy indicates that the co PY06306Ad5 vaccine induces higher protection in CD1 mice (100%, 100%, 86% and93%) compared to the na PY06306 had lower protection (86%, 93%, 86% and71%) for the same Ad5 doses. The study also compared subcutaneous (SC)and intravenous (IV) routes for Ad5 administration. SC route yieldedsimilar protection levels for both na and co PY06306 vaccine (50 and 57%respectively). The IV route for the co PY06306 Ad5 resulted in 100%sterile protection, while the na provided 79%. The IV route for naPY06306 Ad5 yielded 79% sterile protection, while subcutaneous yielded50% protection. Mice groups immunized with a single dose of coPY06306Ad5 induced 93% sterile protection compared to 29% for the naPY06306vaccine. These mice received no DNA vaccine priming. All protectionstudies were performed under animal protocols D02-09 and 14-IDD-13.

Example 12 Human P. falciparum is Immunogenic in Mice

FIG. 13 shows that P. falciparum PFA0205w (E140 ortholog) is immunogenicin mice. Four vaccine reagents were generated for PFA0205w (akaPF3D7_0104100), these are: VR1020 DNA vaccine construct, humanAdenovirus 5 construct, protein expression plasmids pEU-E01-GST, andpEU-E01-His. DNA vaccine and Ad5 were produced in large scale for miceimmunizations. Recombinant proteins were produced in small-scale by thewheat germ cell-free system at NMRC. Both CD1 and BALB/c mice wereimmunized using a variety of prime-boost regimens as shown in FIG. 13.The Ad5 prime and recombinant protein boost was the most immunogenicregimen, inducing IFA titers up to 1:4,000 to both P. falciparum bloodand sporozoite stage parasites. A single dose of PFA0205w Adeovirus 5induce antibodies to parasites. This confirms that PFA0205w as a singledose of recombinant virus (Adenovirus 5) or as a prime-boost with anAd5-protein regimen are viable as vaccine formulations.

Example 13 P. falciparum E140 (PFA0205w) is Immunogenic in Humans

FIG. 14 shows that the P. falciparum E140 (PFA0205w) is immunogenic inhumans. T cells from individual subjects immunized withradiation-attenuated sporozoites (RAS) were able to respond tostimulation with PFA0205w peptide pool (A). The peptide mixturecontained 15 mer overlapping peptides covering most of the N-terminusregion of PFA0205w protein. Since the protein is large, the peptideswere divided into two pools; pool A covering the N-terminus and pool Bfor the C-terminus of the PFA0205w protein. The data in both graphsindicated that the imuunizations with the attenuated sporozoite vaccineinduce both CD4 and CD8 T cells in humans. CD4+and CD8+ T cells play arole in the PFA0205w-induced protection against pre-erythrocyticparasites. There are high levels of P. yoelii E140 responses in thespleen and livers of E140-immunized mice.

Example 14 PFA0205w is Expressed in Schizonts and Localized in theSurface and Cytosol of Sporozoites

The PFA0205w antigen is expressed at both the sporozoite and schizontstages of P. falciparum. The IFA reactivity was obtained using CD1 miceserum generated by priming with PFA0205w Adenovirus 5 and boosting withrecombinant PFA0205w protein. The serum was positive for 36-hour P.falciparum erythrocytic schizonts and negative for early rings andtrophozoites. The subcellular localization of PFA0205w antigen insporozoites was determined by immuno electron microscopy (EM). Theanalysis of micrographs showed that PFA0205w antigen is localized inboth at the surface and in the cytosol of P. falciparum sporozoites.Immuno fluorescence and immuno electron microscopy showed reactivity ofserum from CD1 mice immunized with PFA0205w Adeno 5 and boosted withrecombinant PFA0205w protein. Air-dried IFA slides were made with NF54P. falciparum parasites about 36 hours after invasion of red blood cell.IFA was performed with 1:500 serum dilution and developed with aFITC-labeled goat anti-mouse Ig. For immuno EM, P. falciparumsporozoites-containing salivary glands were isolated from infectedmosquitoes. Fixed glands were embedded, sectioned, mounted on electronmicroscopy grids and stained using same serum and colloidal gold-labeledanti-mouse antibodies. Micrographs confirmed that the PFA0205w antigenis localized in both at the surface and in the cytosol of P. falciparumsporozoites.

Example 15 PVX 081555 (PvE140) is Expressed in P. vivax Sporozoites

FIG. 15 shows that PVX_081555 (PvE140) is expressed in P. vivaxsporozoites. Anopheles dirus mosquitos were fed blood through a membranefeeder from patients infected with P. vivax malaria. Fourteen days afterthe membrane feeding the mosquito salivary glands were extracted from100 mosquitos. The salivary glands were crushed in a microcentrifugetube containing phosphate-buffered-saline with a pestle and to liberatePlasmodium vivax sporozoites. The salivary gland debris-sporozoitemixture was then centrifuged to remove the mosquito salivary glanddebris and the P. vivax sporozoites were transferred from thesupernatant to a new microcentrifuge tube. The extracted P. vivaxsporozoites were counted and 1X106 sporozoites were digested with 1 ugof molecular biology grade trypsin at 37 degrees Celsius for 18 hours.After digestion the trypstic sporozoite peptides were desalted over a C8reversed phase column and lyophilized. The lyophilized tryptic peptideswere subjected to multi-dimensional-protein-identification technology(MudPIT) to identify P. vivax sporozoite proteins that might be vaccinecandidates. Tandem mass spectra generated from P. vivax sporozoites weresearched against a combined Anopheles-Plasmodium vivax protein sequencedatabase using the Sequest algorithm. Output files from the Sequestsearch were loaded into the Scaffold protein viewer. Sequences matchingthe Anopheles proteome were subtracted using the Scaffold program tohighlight proteins that specifically matched the P. vivax proteome.Scaffold software was used to compare the abundance of each of the P.vivax sporozoite proteins identified by MudPIT. Protein abundance wasdefined by the Scaffold “quantitative value” which normalizes theabundance of mass spectra matching a given protein to that protein'smolecular weight. 256 high-confidence P. vivax proteins were identifiedin this MudPIT experiment. P. vivax E140 (PVX_081555) was the 39th mostabundant P. vivax sporozoite protein and the 5th most abundantmembrane-associated protein sequenced. In comparison, the CSP vaccineantigen, that is also associated with the parasite membrane, was the 5thmost abundant protein overall and the most abundant membrane-associatedprotein in the sample. This result illustrates that P. vivax E140 isamong the most abundant membrane-associated proteins in the parasite.E140′s membrane association and abundance therefore make it anexceptional target of the humoral response.

1-13. (canceled)
 14. A method of inducing an immune response againstmalaria in a subject, comprising administering to the subject animmunogenic composition comprising: (a) a recombinant polypeptide;wherein the recombinant polypeptide comprises SEQ ID NO:3, or arecombinant polypeptide that comprises at least 10 contiguous aminoacids of SEQ ID NO:3 and has at least 85% sequence identity with SEQ IDNO:3; (b) a pharmaceutical acceptable carrier; and (c) an adjuvant. 15.The method of claim 14, wherein the recombinant polypeptide comprises atleast 10 contiguous amino acids of SEQ ID NO:3 and has at least 92%sequence identity with SEQ ID NO:3.
 16. The method of claim 14, whereinthe recombinant polypeptide comprises SEQ ID NO:3.
 17. The method claim14, wherein the recombinant polypeptide is an E140 antigen of aPlasmodium falciparum strain.
 18. The method of claim 17, wherein thestrain selected from the group consisting of 3D7, UGT5.1, 7G8, Mali,UGPA, HB3, Santa Lucia, IGH-CR14, FCH/4, NF135/5-C10, Tanzania, FVO, andDd2.
 19. The method of claim 14, wherein the recombinant polypeptide isproduced using an expression vector for prokaryotic or eukaryoticexpression.
 20. The method of claim 19, wherein the expression vector isa plasmid, replicating viral vector, or non-replicating viral vector.21. The method of claim 19, wherein the expression vector is a DNAplasmid, baculovirus, rVSV, SpyVLP, alphavirus replicon, adenovirus,poxvirus, adeno-associated virus, cytomegalovirus, canine distempervirus, yellow fever virus, retrovirus, RNA replicon, DNA replicon,alphavirus replicon particle, Venezuelan Equine Encephalitis virus,Semliki Forest virus, or Sindbis virus.
 22. The method of claim 14,wherein the immunogenic composition is administered as a DNA-basedvaccine platform selected from DNA plasmids or viral systems.
 23. Themethod of claim 22, wherein the viral system is Modified Vaccinia Ankara(MVA) attenuated poxvirus, Vesicular Stomatitis Virus (VSV), or GC46(gorilla adenovirus) virus.
 24. The method of claim 14, wherein theadjuvant is selected from the group consisting of an Army LiposomeFormulation (ALF) derivative, a lipid A derivative, a saponin in QS21, asaponin in 3D-monophosphoryllipid A, lipopolysaccharide (LPS),monophosphoryl lipid A (MPL), 3-O-deacylated monophosphoryl lipid A(3DMPL), acylated monosaccharides, a saponin derivative, solubletriterpene glycosides, Toll-like receptor 4 (TLR4) agonists, montanideISA51, montanide ISA720, immunostimulatory oligonucleotides, andimidazoquinolines.
 25. The method of claim 14, wherein the ALFderivative is selected from the group consisting of ALF, ALF plusaluminum (ALF A), and ALF plus QS21 (ALFA).
 26. The method of claim 14,wherein the saponin derivative is selected from the group consisting ofQuil-A, Immune stimulating complexes (ISCOM), QS-21, AS02, and ASO1. 27.The method of claim 14, wherein the immunogenic composition is asolution, a suspension, or an emulsion.
 28. The method of claim 14,wherein the subject is a human.
 29. The method of claim 14, wherein thecomposition is administered orally, systemically, parenterally,topically, mucosally, intramuscularly, intravenously, intraperitoneally,intradermally, subcutaneously, intranasally, intravaginally,intrarectally, transdermally, sublingually, via an aerosol route, or viainhalation.
 30. The method of claim 14, wherein the composition isformulated as an ampule, a pre-filled syringe, a small volume infusioncontainer, or in multi-dose containers with an added preservative. 31.The method of claim 30, wherein the composition is formulated as apre-filled syringe.
 32. A method of inducing an immune response againstmalaria in a subject, comprising administering to the subject animmunogenic composition comprising: (a) a recombinant polypeptide;wherein the recombinant polypeptide comprises SEQ ID NO:3, or arecombinant polypeptide that comprises at least 10 contiguous aminoacids of SEQ ID NO:3 and has at least 85% sequence identity with SEQ IDNO:3; (b) a pharmaceutical acceptable carrier; and (c) an adjuvant;wherein the immunogenic composition further comprises one or moreadditional recombinant polypeptides, wherein the one or more additionalrecombinant polypeptides comprise an amino acid sequence selected fromthe group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, andSEQ ID NO: 9, or a recombinant polypeptide that comprises at least 10contiguous amino acids of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, andSEQ ID NO: 9 and has at least 85% sequence identity with one of SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:
 9. 33. The method ofclaim 32, wherein the recombinant polypeptide comprises SEQ ID NO:3. 34.The method of claim 33, wherein the one or more additional recombinantpolypeptides comprise SEQ ID NO:
 6. 35. The method of claim 33, whereinthe one or more additional recombinant polypeptides comprise SEQ ID NO:7.
 36. The method of claim 33, wherein the one or more additionalrecombinant polypeptides comprise SEQ ID NO:
 8. 37. The method of claim33, wherein the one or more additional recombinant polypeptides compriseSEQ ID NO:
 9. 38. The method of claim 32, wherein the recombinantpolypeptide is produced using an expression vector for prokaryotic oreukaryotic expression.
 39. The method of claim 38, wherein theexpression vector is a plasmid, replicating viral vector, ornon-replicating viral vector.
 40. The method of claim 38, wherein theexpression vector is a DNA plasmid, baculovirus, rVSV, SpyVLP,alphavirus replicon, adenovirus, poxvirus, adeno-associated virus,cytomegalovirus, canine distemper virus, yellow fever virus, retrovirus,RNA replicon, DNA replicon, alphavirus replicon particle, VenezuelanEquine Encephalitis virus, Semliki Forest virus, or Sindbis virus. 41.The method of claim 32, wherein the immunogenic composition isadministered as a DNA-based vaccine platform selected from DNA plasmidsor viral systems.
 42. The method of claim 41, wherein the viral systemis Modified Vaccinia Ankara (MVA) attenuated poxvirus, VesicularStomatitis Virus (VSV), or GC46 (gorilla adenovirus) virus.